[20] ONE-STEP SYNTHESIS OF HIGH FLUORESCENT BSA QUANTUM DOTS
Vijay Bhooshan Kumar1
1 Bar Ilan Institute for Nanotechnology and Advanced Materials; Bina, Department of Chemistry, Bar Ilan University
One-Step Synthesis of high fluorescent BSA quantum dots
Vijay Bhooshan Kumara, Aharon Gedankena, b*
aDepartment of Chemistry and Bar-Ilan Institute for Nanotechnology & Advanced Materials, Bar Ilan University, Ramat-Gan 52900, Israel
bNational Cheng Kung University, Department of Materials Science & Engineering, Tainan 70101, Taiwan
*Corresponding author email: [email protected]
Fax: 972-3-7384053; Tel: 972-3-5318315
Abstract:
In the current work, we present a new general facile synthesis of BSA (bovine serum albumin) quantum dots from BSA polymer, using hydrothermal reaction. The as-prepared BSA QDs exhibited high quantum yield, high photostabiliy and colloidal stability, and high functionalization efficiency. Interestingly, a high Quantum yield (ca. 40%) was observed with the help of various spectroscopy techniques. However, the advantage of these particles are nontoxic and long stability for the biological applications (in vitro and in vivo) as well as the low cost. Importantly, with high physiological stability, excellent biocompatibility, and homogenous distribution of BSA QD suspension was used to obtain a high contrast bio-imaging and life cell imaging.
[48] THE DYNAMIC PROPERTIES OF A DNA ORIGAMI POLYMER STRUCTURE
Efrat Roth1
1 Bar- Ilan University; Bar- Ilan University
The Dynamic Properties of a DNA Origami Polymer Structure
Efrat Roth and Yuval Garini
Abstract:
DNA origami is an enabling technique that has been developed during the last decade. Using this technique, one can design almost any kind of form and 3-dimensional structure and build many copies of it in a nanometer scale in a very exact way. Little has been studied about dynamic properties of systems constructed by DNA origami. A polymer-like structure will be built using the DNA origami method, which is a new concept. The polymer consists of rigid rods connected to each other by short double-stranded DNA. The structure allows each rod a wide degree of freedom to move so that it mimics a polymer that may be described by the freely joint chain or other polymer models. The Polymer-like structures will be measured by using tethered particle motion (TPM) method and atonic force microscopy. Both the polymer conformations and dynamics will be studies and it will allow us to check whether the polymer’s properties match the expectations according to the model and the previous measurements of dsDNA. The synthesized polymer-like structures can be used later on for further biophysical studies, as building blocks for biosensors and even for biomedical applications.
[52] DESIGN OF POLY(STYRYL BISPHOSPHONATE) NANOPARTICLES GRAFTED ONTO POLYPROPYLENE FILMS FOR INHIBITION OF BIOFILM FORMATION
Hanna Adelman-Steinmetz1, Safra Rudnick-Glick2, Michal Natan3, Ehud Banin4, shlomo Margel5
1 The Institute of Nanotechnology and Advanced Materials ; Department of Chemistry
2 Department of Chemistry ; The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University,
3 The Institute for Advanced Materials and Nanotechnology; Bar Ilan University
4 The Mina and Everard Goodman Faculty of Life Sciences; The Institute for Advanced Materials and Nanotechnology
5 Biu; Department of Chemistry & The Institute of Nanotechnology and Advanced Materials
In the past decades bacterial biofilms have been intensively studied for medical and industrial applications. Biofilms cause hazardous contaminations since they have a high resistance to antibiotics and hostile environments. Their unique structure consists of bacterial aggregates which attached to the surface. Ca2+ ions play an important role in the formation of biofilms and impact their stability, architecture, viscosity and strength. Bisphosphonates are a stable chemical analog of the inorganic pyrophosphate and exhibit a high affinity to Ca2+ ions. Bisphosphonates may inhibit the formation of biofilms by acting as sequestering agents for Ca2+ ions. In this study we present a new approach for antibacterial coating, based on bisphosphonates, on polymeric materials while maintaining the mechanical properties. Polypropylene films were treated by O2 plasma to form the desired conjugated-hydroperoxide groups which used as initiator for the graft polymerization of the novel styryl bisphosphonate monomer. The produced bisphosphonate surface coating was confirmed by surface measurements including XPS, AFM, ATR-IR and contact angle. A significant inhibition of the biofilm formation was achieved for both gram-negative (Escherichia coli) and gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) bacteria.
[74] ENHANCED ACTIVITY OF IMMOBILIZED PEPSIN NANOPARTICLES COATED ON SOLID SUBSTRATES COMPARED TO FREE PEPSIN
David Meridor1, Aharon Gedanken2
1 Bar-Ilan University; Bar-Ilan University
2 Department of Chemistry, Institute of Nanotechnology and Advanced Materials, Bar-Ilan University; Bina
Enhanced activity of immobilized pepsin nanoparticles coated on solid substrates compared to free pepsin
Pepsin, an aspartic peptidase, is used for a variety of applications in industry. Pepsin is sensible to changes in the environment and requires improvement in its catalytic efficiency. Immobilization of pepsin onto solid supports can offer advantages such as easier separation of the enzyme from the product and the possibility of repetitive use of a single batch of enzyme.
In the present work, nanoparticles of pepsin were generated in an aqueous solution using high intensity ultrasound, and were subsequently immobilized on low-density polyethylene (PE) films, or on polycarbonate (PC) plates, or on microscope glass slides. The leaching properties, and the catalytic activity of the immobilized enzyme on the three surfaces were compared. Catalytic activities of pepsin deposited onto the three surfaces as well as free pepsin, without sonication, and free pepsin NPs were compared at various pH levels and temperatures. Compared to native pepsin, pepsin coated onto PE showed the best catalytic activity in all the examined parameters. Pepsin immobilized on glass exhibited better activity than the native enzyme, especially at high temperatures. Enzyme activity of pepsin immobilized on PC was no better than native enzyme activity at all temperatures at pH 2, and only over a narrow pH range at 37 oC was the activity improved over the native enzyme. A remarkable observation is that immobilized pepsin on all the surfaces was still active to some extent at pH 7, while free pepsin was completely inactive. The kinetic parameters, Km and Vmax were also calculated and compared for all the samples. Relative to free pepsin, pepsin coated PE showed the greatest improvement in kinetic parameters (Km= 15 g/L, Vmax= 719 U/mg versus Km= 12.6 g/L and Vmax= 787 U/mg, respectively), whereas pepsin coated on PC exhibited the most unfavorable kinetic parameters (Km= 18 g/L, Vmax= 685 U/mg).
Keywords:
Sonochemistry, pepsin nanoparticles, immobilization, enzyme activity
[81] MAGNETIC NANOPARTICLES FOR MANIPULATING NEURONAL GROWTH
Michal Marcus1, Moshe Karni1, Koby Baranes1, Noa Alon1, Itay Levy2, Tony Yamin3, shlomo Margel2, Amos Sharoni3, Orit Shefi1
1 Biu; Faculty of Engineering & The Institute of Nanotechnology and Advanced Materials
2 Biu; Department of Chemistry & The Institute of Nanotechnology and Advanced Materials
3 Biu; Department of Physics & The Institute of Nanotechnology and Advanced Materials
The ability to manipulate neuronal organization and growth has extensive implications in neuronal regeneration and tissue engineering. In the present study we use magnetic nanoparticles (maghemite, γ-Fe2O3) as mediators to apply physical forces locally and as carriers of neuronal growth factors. We use these nano-complexes in order to locate cells, promote neuronal growth and affect growth orientation. We designed and generated magnetic fields with controlled magnetic flux densities at multiple scales of size and strength. We fabricated a unique device embedded with micro-patterned pads that can be magnetized selectively. We incubated PC12 cells and primary neurons, in medium enriched with iron oxide nanoparticles conjugated to fluorescent tag. Both types of cells uptake the nanoparticles and turned sensitive to the magnetic stimulation with no cytotoxic effect. Plating PC12 cells atop the micro-patterned device has led to an organized network of clusters of cells. Currently we are mathematically modelling nanoparticles uptake by cells and the organization of magnetized cells in response to various external magnetic fields. In addition, we found that covalent conjugation of the magnetic nanoparticles to nerve growth factor (NGF) which is a critical component in nerve tissue development and repair enhanced the typical effect of NGF. Morphometric and molecular measurements revealed that treatment with the nanoparticle-NGF complex leads to a promoted differentiation progression and to more complex dendritic trees. Stability and signaling pathway assays suggest conjugation to NPs as a method to extend the half-life of NGF, thereby increasing its availability and efficiency. Our study presents an emerging magneto-chemical method for the manipulation of neuronal migration and growth opening new directions in non-invasive neuronal repair.
[93] DIRECTING NEURONAL GROWTH IN 3D COLLAGEN HYDROGELS
Mereav Antman-Passig1, Orit Shefi2
1 Faculty of Engineering, Bar-Ilan University; Insititue for Nanotechnology and Advanced Materials, Bar-Ilan University
2 Biu; Faculty of Engineering & The Institute of Nanotechnology and Advanced Materials
The ability to manipulate and direct neuronal growth has great importance in the field of tissue engineering, both for neuronal repair and potential medical devices. Since mammalian neurons have limited regeneration abilities creating scaffolds for enhanced regeneration is beneficial. Moreover, guiding and directing neuronal outgrowth can enhance neuronal repair and recovery. Previous studies, including in our lab examined nanoparticles for enhancing neuronal regeneration. Here, we designed a 3D collagen gel-scaffold for neuronal cultures. We further modulated the gel system to create alignment of collagen fibers for directing neuronal growth using nanoparticles.
A collagen hydrogel system was chosen as a 3D ECM analog to best mimic the natural environment of cells. The gels mechanical properties were examined and tuned to achieve desired properties similar to nervous tissue. We compared the neuronal growth in 3D to a 2D model and showed that neurons grown in 3D collagen gels develop significantly longer dendritic trees and neurites. To manipulate neuronal growth we developed a method to align collagen fiber matrix by incorporating magnetic nanoparticles within gels, and exposing the gel to an external magnetic field We showed fiber directionality by analysis of light microscope images via Fast Fourier transform (FFT) and by SEM imaging. We grew neurons in aligned gels for 7 days and followed regeneration process of single cells for up to 7 days. For this purpose we used both primary leech (Hirudo medicinale) neuronal culture, and PC12 as a mammalian analog. Using a designed Matlab script we evaluated cellular direction of growth and compared it to collagen matrix orientation. We further measured morphometric parameters of neuronal growth. Using aligned gels we’ve elongated and directed neuronal growth coinciding with collagen matrix orientation. We also found aligned gels initiate neurite growth patterns similar to growth in 3D control gel.
[94] NANO STRUCTURED POROUS SILICON CHIPS FOR NEURONAL DIFFERENTIATION
Neta Zilony1, Orit Shefi2, michal Rosenberg3, Esty Segal3, Liran Holtzman4
1 Faculty of Engineering and Institute of Nanotechnology and Advanced Materials; Bar Ilan
2 Biu; Faculty of Engineering & The Institute of Nanotechnology and Advanced Materials
3 Department of Biotechnology and Food Engineering; Technion
4 Department of Biotechnology and Food Engineering,; Technion
Nano Structured Porous Silicon Chips for Neuronal Differentiation
Zilony N.1, Rosenberg M.2, Holtzman L.2, Segal E.2 and Shefi O.1
1Faculty of Engineering, Bar-Ilan University Ramat-Gan, Israel
2Department of Biotechnology and Food Engineering, Technion, Haifa, Israel
Nerve growth factor (NGF) is a well characterized protein and an essential contributor to neuronal differentiation. NGF has shown high pharmacological potential in several models of neurodegenerative diseases. However, growth factors undergo rapid degradation which leads to a short biological half-life. Therefore, its effectiveness in therapeutics is limited. Recent work showed an enhancement in the NGF activity due to covalent conjugation of NGF to iron oxide nanoparticles. In our study, we develop a delivery system, composed of porous Silicon (PSi) chips, that allows sustained release of NGF. Nanostructured PSi is characterized by several particularly appealing tunable properties predestining it for design of drug delivery systems, including high surface area, biocompatibility and ability to degrade completely in physiological environment. Different PSi nanostructures were fabricated by anodic electrochemical etching of single-crystalline Si wafers and the synthesis conditions were adjusted to allow efficient loading of NGF by physical adsorption. In order to study the effect of the combined complex, we used a neuronal like cells model (PC12 cells). NGF is an essential factor for the maintenance and differentiation of PC12 cells in culture. The NGF-chips were added to PC12 cells in culture and the cell began to differentiate. The differentiation was determined by the neurites number, the neurites total length and by molecular markers. The culture was sustained with the NGF-chips up to 14 days. Our work aims to develop new PSi-based carriers for the controlled release of NGF. We demonstrate that NGF entrapment within the PSi allows for its sustained delivery to promote differentiation of PC12 cells without any need of external supplement. Therefore, this novel system will be able to be used in in vivo trails.
[108] PDOTS NANOPARTICLES LOAD PHOTOSENSITIZERS AND ENHANCE EFFICIENTLY THEIR PHOTODYNAMIC EFFECT BY FRET
Elina Haimov1, Hana Weitman1, Debby Ickowicz2, Zvi Malik2, Benjamin Ehrenberg1
1 Department of Physics; Institute of Nanotechnology and Advanced Materials
2 Faculty of Life Sciences; Bar Ilan University
A new type of nanoparticles, Pdots, and a new methodology of photosensitization are developed to achieve a more efficient photodynamic effect in aqueous solutions and in cells. Pdots are nano-sized particles, composed of conjugated chromophoric polymers coated with PEGylated phospholipids. They exhibit good aqueous colloidal properties, a broad absorption band and a strong and narrow emission band. We show that these characteristics improve biological photosensitization, which is employed in photodynamic therapy of cancer. Pdots nanoparticles load amphiphilic photosensitizers such as Rose Bengal with a high affinity, into the amphiphilic coating, without necessitating covalent attachment. At this close contact, very efficient fluorescence resonance energy transfer (FRET) occurs between the Pdot donor and the sensitizer acceptor. The Pdots serve as broad-band collectors of light, which is funneled, via FRET, to the photosensitizer. Therefore, FRET from them can additively assist to the activity of the acceptor’s energy. The efficient FRET mechanism, strong uptake of the Pdot-sensitizer dyads by MCF-7 adenocarcinoma cells and their enhanced photosensitized killing are demonstrated.
[128] A NOVEL APPROACH TO THE DECORATION OF PARYLENE C USING FUNCTIONAL SILICA-TRICLOSAN CO-POLYMERS NANOPARTICLES
Rina Binyamini1
1 Bar-Ilan; Biotechnology Chemistry
A Novel Approach to the Decoration of Parylene C Using Functional Silica-Triclosan co-polymers Nanoparticles
Rina B. Binyaminy a, Edith Laux b, Herbert Keppner b, Ehud Banin c
and Jean-Paul Lellouche a*
a Department of Chemistry, Faculty of Exact Sciences, Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900, Israel
b Haute Ecole Arc Ingénierie (HES-SO), CH-2300 La Chaux-de-Fonds, Switzerland
c The Mina and Everard Goodman Faculty of Life Science, Institute for Nanotechnology & Advanced Materials, Bar-Ilan University, Ramat-Gan 52900, Israel
*Corresponding author, Prof. Jean-Paul Lellouche. Email: [email protected]
Both pure silica (SiO2) nanoparticles (SNPs) and functionalized hybrid triclosan/silica nanocomposites (T-SNC) were deposited onto non-functional Parylene C films using a novel, readily executed, one-step decoration method. Unlike previously known methods, this functionalization method of Parylene C films required neither a binding agent nor sophisticated equipment/devices. The SiO2-based NPs anchored on Parylene C substrates were formed via a common base-catalyzed hydrolytic sol-gel method. Regarding the mechanism, it has been assumed that the SiO2 phase precursor (Si(OEt)4, tetraethyl orthosilicate, (TEOS) was first adsorbed and 2D polymerized onto the parylene C film due to hydrophobic interactions that served as an anchor mechanism for further NP growth. This assumption was investigated by comparing thermal behaviors (measured by differential scanning calorimetry, DSC) of parylene C coatings before and after the following specific surface treatment, i.e., (i) first parylene C coating incubation with TEOS followed by (ii) SNP formation and growth from such a TEOS-modified coating surface. Following the same procedure, hybrid thiophene-containing H-SiO2-Tricl NPs were also successfully grown from the surface of a TEOS-modified parylene C film and characterized using high resolution scanning electron microscopy (HR-SEM) and X-ray photoelectron spectroscopy (XPS). In order to obtain deeper insight into the overall functionalization process, the similar hybrid H-SiO2-Tricl NPs that formed in the bulk contacting medium were also isolated and fully characterized for comparison needs. Biological experiments were done as well.
[171] SURFACE MODIFICATION OF CELLULOSE NANOCRYSTALS WITH GLYCIDYL METHACRYLATE FOR UV-CROSSLINKING
Yuval Nevo1, Yifeng Cao2
1 The Hebrew University of Jerusalem; Robert H. Smith Faculty of Agriculture, Food and Environment
2 The Hebrew University; Robert H. Smith Faculty of Agriculture, Food and Environment
Cellulose nanocrystals (CNCs) are considered to be promising natural components for composite reinforcement, coatings, light-weight foams and hydrogels. Here, CNCs were modified with glycidyl methacrylate (GMA) to introduce C=C groups that can be UV-crosslinked to give CNC based hydrogels and aerogels. The effects of various parameters (molar ratio of GMA to hydroxyl groups, different catalysts, reaction time, temperature, etc.) on the degree of substitution were investigated in order to optimize the reaction conditions. The GMA modified CNCs (GMA-m-CNCs) were characterized by ATR-FTIR, solid state 13C-NMR, POM and XRD. Hydrogels prepared using CNCs modified using the optimized reaction conditions were stable in water and other organic solvents. In addition, methacrylated CNCs were introduced as nanoreinforcing agents inside acrylamide matrices, resulting in enhanced mechanical properties and better crosslinking density of the polymer.
[255] REALIZING ULTRASENSITIVE LIVE CELL ATOMIC FORCE IMAGING WITH LARGE FORCE CONSTANT FUNCTIONAL GLASS PROBES
Aaron Brahami1, Efrat Zlotkin-Rivkin2, Benjamin Aroeti2, Aaron Lewis1
1 Department of Applied Physics; The Selim and Rachel Benin School of Computer Science and Engineering
2 Hebrew University of Jerusalem; Silberman Life Sciences Institute
Since the invention of atomic force microscopy (AFM) in 1986 live cell imaging has gradually progressed inspite of fundamental limitations in generally applied laser beam deflection (LBD) force sensing. This has been achieved by developing algorithmically based protocols to quantitatively delineate the interactions of AFM probes with cell surfaces. A recent effort was the application of a relatively recent algorithm to image fine cellular protrusions or microvilli, a previously unachievable goal [1]. For this advance ultrasoft silicon probes with cantilever force constants of 0.0611N/m were required. A significant next step would be to implement the same ultrasensitive live cell imaging with an important class of large force constant (1-10N/m) functional glass probes for applications such as near-field scanning optical microscopy (NSOM), AFM sensing with patch clamping pipettes [1], scanning electrochemical microscopy etc. In the presentation this next step in live cell imaging is described, with considerable import for scanned probe imaging of live cells.
[1]. H. Schillersa, I. Medalsyb, S. Hub, A. L. Sladeb and J. E. Sha
[267] SYNTHETIC GENE CIRCUITS INTEGRATED ON A CHIP
Alexandra Tayar1, Eyal Karzbrun2
1 Weizmann Institute of Science; M&i Perlamann 414
2 Weizmann Institute of Science; Department of Molecular Genetics
Synthetic gene circuits integrated on a chip
Alexandra M. Tayar1, Eyal Karzbrun1, Vincent Noireaux2, Roy H. Bar-Ziv1, 3
1Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel, 76100
2Department of Physics, University of Minnesota, Minneapolis, Minnesota, 55455, USA
Abstract
Gene circuits regulate fundamental cellular functions, from macroscopic spatiotemporal patterns in development to assembly of structures and machines at the nanoscale. Integration of synthetic transcription-translation circuits into solid-state devices, with control of local RNA and protein synthesis, would enable programmable devices and assembly lines for applications in nanobiotechnology. We created a diffusive system of confined localized gene expression sources and sinks, which drive the emergence of rich dynamic states of patterned gene circuits. Proteins are synthesized from crowded DNA brushes (source) patterned in silicon-fabricated 2D compartments about a micron deep. Steep steady-state linear protein concentration gradients form along a capillary connecting the DNA compartments to a flow reservoir (sink). We observe steady-state expression, pulses, and oscillations, with protein levels and period times linear in the capillary length, thereby establishing a geometric means to regulate circuits in the device. Transcription regulation in the 100nm thick DNA brush is directly observed in real-time. More than 80 circuits were integrated in a single device, which in principle could be scaled up to thousands of micron-size compartments using current fabrication techniques1,2.
1. Karzbrun E, Tayar AM, Noireaux V, Bar-Ziv RH. Programmable on-chip DNA compartments as artificial cells. Science. 2014;345(6198):829–832. doi:10.1126/science.1255550.
2. Tayar AM, Karzbrun E, Noireaux V, Bar-Ziv RH. Propagating gene expression fronts in a one-dimensional coupled system of artificial cells. Nat Phys. 2015;advance on. doi:10.1038/nphys3469.
[278] LIGHT ACTIVATED HYBRID NANOPARTICLES FOR BIO-MEDICAL APPLICATIONS
nir waiskopf1, Yuval Ben Shahar2, Hermona Soreq3, Uri Banin4
1 Institute of Chemistry and the Center for Nanoscience and Nanotechnology; Dept. of Biological Chemistry, The Hebrew University of Jerusalem
2 The Institute of Chemistry and Center for Nanoscience and Nanotechnology; The Hebrew University of Jerusalem
3 Dept. of Biological Chemistry; The Hebrew University of Jerusalem
4 Institute of Chemistry and the Center for Nanoscience and Nanotechnology; The Hebrew University of Jerusalem
Hydrogen peroxide (H2O2) can cause significant damage in biological systems, culminating in cell death. However, local and tunable control of H2O2 production can be of utmost importance. For example, the enzyme thyroid peroxidase uses H2O2 for the production of the thyroid hormones. Its depletion leads to hypothyroidism, resulting in fatigue, joint and muscle pain, depression and more. At the translational research level, horseradish peroxidase (HRP) and hydrogen peroxide are employed in many detection and quantification methods where the introduction of hydrogen peroxide at high spatial-temporal resolution offers an added value.
Recent developments and progress in the synthesis of semiconductor-metal hybrid nanoparticles (HNPs) allow unprecedented control over the composition and structural properties of these hybrid systems, promoting the ability to tune its chemical and physical characteristics. Such control opens new opportunities for utilizing HNPs in diverse applications. A unique synergistic property that arises from the semiconductor-metal interface is an efficient spatial charge separation. This results in efficient functioning and photo-catalysis in redox reactions, including hydrogen generation by water splitting, and photo-degradation of organic contaminants.
Here we present HNP-mediated formation of radicals and peroxides amenable for use in the controlled local modulation of biological systems, with important research and bio-medical utilities. As a model system we show potentiation of HRP activity and inhibition of cholinesterase activities by light stimulation of CdSe/CdS-Au HNPs, as well as the option to add supportive enzymes (e.g superoxide dismutase (SOD)) or molecules (hole acceptors) which increase the efficiency and specificity of the system. We further demonstrate the advantages in the use of HNPs over bare semiconductor nanoparticles and the context-dependence of these capacities on the HNP properties.
The ability to control radicals and peroxides formation in a high spatio–temporal manner using HNPs excitation can deepen our understanding of the corresponding biological processes, improve the available tools for light modulation of enzymatic activities and open new avenues for developing novel treatments for diverse diseases.
[417] PEPTIDE INDUCED PLASMONIC CIRCULAR DICHROISM AS A SENSITIVE PROBE OF PEPTIDE CONFORMATION
Tatyana Levi Belenkova1, Gil Markovich2
1 School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences; Tel Aviv University
2 School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences; Tel-Aviv University
Chirality is an important theme in bio-molecular and organic chemistry due to its key role played in biological processes and in determining pharmacological, pharmacokinetic and toxic features of various molecules. When bio-molecules are assembled on metal nanostructures they can impart optical activity to the nanomaterial in the form of plasmonic circular dichroism (CD) signal in the visible spectral region.
In our study we explore plasmonic CD signal induced by short polyproline peptides adsorbed to silver nanocubes (NCs). Interestingly, the CD induction occurs for one specific plasmon resonance mode and not in other modes. We show that the plasmonic CD signal inverts its polarity when the adsorbed molecule orientation is inverted. This is due to a change in the orientation of transition dipole moments of chromophores in the peptide chain (mainly carbonyl groups) with respect to the metal surface.
This work demonstrates that CD induction by chiral molecules in noble metal nanostructures with well defined geometries may be used as a sensitive conformational probe and provide information about the adsorbed configuration of the molecules. Such molecule-NC assemblies might be useful for chiral separations.
[431] EXCITON SUPER TRANSFER IN BIOLOGICAL NANO-WIRES
Ido Eisenberg1
1 Hebrew University; Hebrew University
Exciton super transfer in biological nano-wires
Antenna complexes of photosynthetic cyanobacteria poses superior excitation transfer efficiency at room temperature. In this study we explored how to control and use these properties in order to get an efficient nano to micro scale energy transmission. The energy transfer is examined using Phycocyanin trimers that are modified and dried on several substrates. Results show ordering of the proteins using two different methods. One method is adding salts to the solution which makes them arranged in orthogonal dendrites. The second method is filling micro-trenches by spin-coating. Using this method we achieve bundles of nano-wires of Phycocyanin. We believe that during the drying process the proteins arrange in super-molecular organizations mimicking the native proteins. Optical measurements indicate large distance of ~1μm size excitation transport mechanisms. Time resolved measurements show that organized structures exhibit shorter exciton life-time than native proteins. Such structures may serve as a nano-metric energy transmission lines, and may be used to couple light to nano-devices.
[432] SINGLE MOLECULE FLUORESCENT ASSAY FOR QUANTIFICATION OF DNA TANDEM REPEATS
assaf grunwald1
1 Tau; School for Chemistry
SINGLE MOLECULE FLUORESCENT ASSAY FOR QUANTIFICATION OF DNA TANDEM REPEATS
Assaf Grunwald a, , ElmarWeinhold b, Fredrik Westerlund C, Yuval Ebenstein a
aDepartment of Chemical Physics, School of Chemistry, Tel Aviv University, Tel Aviv, Israel, bInstitute of Organic Chemistry, RWTH Aachen University, Aachen, German ,cDepartment of Chemical and Biological Engineering, Chalmers University of Technology, Goteborg, Sweden
[email protected]
Studies and quantification of DNA repetitive elements is a major stumbling block for next generation sequencing methods, due to difficulties in assembly of identical short reads. Other methods used today to assess repeats offer relative, averaged and inaccurate results. Thus, these regions, which occupy almost 50% of the human genome(ref), are still poorly characterized. Accurate quantification of genetic repeat regions is especially important for the diagnosis of some genetic disease, characterized by an impaired number of DNA repeat units, such as FSHD, the third most common muscular dystrophy.
Optical mapping of DNA enables direct visualization of individually stretched DNA molecules. DNA stretching is enabled by forcing it into a nano-channels array using an electric field. This concept can be harnessed for repeat quantification if a distinct fluorescent signal could be encoded on each repeat unit, allowing direct physical counting of repeats.
We applied this approach to characterize BAC DNA containing the FSHD disease associated repeat array, and its surrounding DNA region. The DNA methyltransferase M.TaqI was used to generate a single fluorescent label on each repeat unit, enabling direct quantification of the repeat array. The same labeling reaction generates a specific labeling pattern on the DNA sequence surrounding the repeat, allowing its identification by measurement of the fluorescent amplitude modulations along the stretched DNA and using cross correlation analysis between the experimental and theoretical fluorescent modulation plots.
We demonstrate initial results towards establishing this assay as a clinical platform for FSHD studies. This would allow rapid detection, from relatively low amounts of DNA, hopefully enabling efficient diagnosis and studies of the disease.
[450] TUNING CHEMILUMINESCENCE OF A LUMINOL FLOW BY PLASMONIC NANOPARTICLES
Alina Karabchevsky1, Ali Mosayyebi2, Alexey Kavokin3
1 Ben-Gurion University of the Negev; 84105
2 2optoelectronics Research Centre University of Southampton; University of Southampton
3 3department of Physics and Astronomy, University of Southampton, So17 1bj; Spin Optics Laboratory, St-Petersburg State University, 1, Ulianovskaya, 198504
Chemiluminescence is a fascinating optical effect that finds its use in various application areas: from forensic science to industrial bio-chemistry. Luminol is a chemical that exhibits chemiluminescence with emitted blue glow light. About five decades ago, luminol was used, for the first time, to analyze a crime scene in Germany. Since then, it became a very popular tool of criminology as it allows for revealing blood stains. We discover a giant increase of the intensity of chemiluminescence of a luminol flow (Figure 1) and a dramatic modification of its spectral shape in the presence of metallic nanoparticles [1]. We observed that pumping gold and silver nanoparticles into a microfluidic device fabricated in polydimethylsiloxane prolongs the glow time of luminol. We demonstrate that the intensity of chemiluminescence in the presence of nanospheres is dependent on the position along the microfluidic serpentine channel. We show that the enhancement factor can be controlled by the nanoparticle size and material. Spectrally, emission peak of luminol overlaps with the absorption band of nanospheres (Figure 1). This maximizes the effect of confined plasmons on the optical density of states in the vicinity of the luminol emission peak. This observations interpreted in terms of the Purcell effect mediated by nano-plasmons is an essential step toward the development of microfluidic chips with gain media. Practical implementation of the discovered effect includes improving detection limits of chemiluminescence for the forensic science, research in biology and chemistry, and for a number of commercial applications.
Figure 1 (a) Experimental images of the studied serpentine taken by a CCD camera. Luminol has been injected with the flow rate of 0.35 L/sec with silver nanoparticles of rSNP=30nm (left) to be compared with reference signal detected in the absence of nanoparticles (right). (b) Intensity over a cross section of images shown in (a). Serpentine arms under investigation are labeled by roman numerals. Note: the subscript SNP abbreviates silver nanoparticles.
[479] ASSEMBLY OF SILK-CBD NANOFIBERS, MIMICKING SPIDER SILK HIERARCHICAL STRUCTURE
zvi shtein1, Oded Shoseyov1
1 The Hebrew University of Jerusalem; Robert H. Smith Faculty of Agriculture, Food and the Environment
Assembly of Silk-CBD Nanofibers, Mimicking Spider Silk
Hierarchical Structure
Biopolymer research has focused in recent years on fibrous proteins due to their unique mechanical properties. Examples include collagen, elastin, silk worm silk, spider dragline silk and resilin. These proteins are distinguished by their repetitive amino acid sequences that confer impressive mechanical properties, such as strength and flexibility.
Spider silk proteins form intrinsic composites dictated from their unique molecular structure that combine highly crystalline domains embedded in amorphous domains resulting in fibers that combine strength and elasticity. In spite of the attractive mechanical properties of silk fibers, spiders produce silk in small quantities, and their territorial behavior prevents large amounts from being raised for farming silk. Consequently isolation of large amounts of silk from spiders is not feasible and these materials must be produced by recombinant protein technologies.
In the present study, we examined the hypothesis that the dimerization of cellulose binding domain (CBD) can direct the ordered assembly of silk proteins into supermolecular fibrillar structures. Nanofiber formation, in the absence of the nonrepetitive N and C termini, is possible due to the novel cellulose binding capability of the recombinant silk-CBD protein.
The fusion spider silk-CBD gene, consists of a synthetic 15 monomer long spider silk gene based on a monomer consensus derived from the native sequence of MaSp1 of Nephila clavipes fused to the 5′ of family III CBD originated from Clostridium cellulovorans, was successfully expressed in E.coli and purified on Ni-NTA under native conditions. Based on the silk-CBD assembly model, suggesting CBD induces spider silk molecular order and can function as the non-repetitive C-termini of MaSp1 protein, we further investigated the ability of CBD to form dimers which is thought to give molecular alignment to silk proteins in the nanofiber formation process. We show that sonication and buffer conditions encourages the self-assembly of nanofibers.
[489] PROGRAMMABLE SELF-ASSEMBLY OF PROTEIN NANO MACHINES ON SILICON CHIPS
Ohad Vonshak1, Shirley Daube1, Roy Bar-Ziv1
1 Weizmann Institute of Science; Department of Materials and Interfaces
Programmable self-assembly of protein nano machines on silicon chips
Ohad Vonshak, Shirley S. Daube, Roy H. Bar-Ziv
Department of Materials and Interfaces
Weizmann Institute of Science
The bottom up design and fabrication of man-made self-assembled machines at the nano scale could be advanced by looking for inspirations in nature. Biological systems operate as an intricate network of association and dissociation reactions run by large macromolecular machines. From structural assemblies that serve as vehicles of genetic materials such as T4 bacteriophage (figure), to multi protein RNA complexes that catalyze complicated biochemical reactions such as the ribosome, these biological nano-machines are self-assembled in vivo in an orchestrated assembly line, avoiding non-specific interactions with thousands of surrounding cytoplasmic proteins and RNA molecules. Could we delineate key mechanistic steps that are required to realize specific, reproducible and efficient nano-machine self-assembly?
Towards mimicking in vitro self-assembly of biological nano-machines, we have devised a methodology to synthesize in cell-free extracts protein and RNA molecules on a silicon chip (figure). The chip can be programed in space and time to dictate assembly of the protein and RNA building blocks in to macromolecular nano-machines. We are using surface-bound genes as the source of assembly components and antibody traps as the localization site for assembled complexes. In a multi-well silicon chip, the surface is patterned by UV light to spatially resolve genes and traps, each well with its own pattern. The genetic program in each well specifies which proteins would be destined to fluorescent labeling or for antibody trapping, respectively. Preliminary results demonstrate that proteins can be synthesized in a cell-free extract and reproducibly form specific multi-protein complexes on the silicon chip. Significantly, assembly outcome may be tunable by the relative geometrical arrangement of genes and traps.
[499] NANOCHANNEL ARRAYS FOR EPIGENETIC MAPPING OF HUMAN CHROMOSOMES
Tslil Gabrieli1, Yuval Ebenstein2
1 Tel Aviv University; Tel Aviv University
2 School of Chemistry; Tel Aviv University
NANOCHANNEL ARRAYS FOR
EPIGENETIC MAPPING OF HUMAN CHROMOSOMES
Tslil Gabrieli, Yael Michaeli, Tamar Shahal, Hila Sharim, Yuval Ebenstein
5-hydroxymethyl-cytosine (5hmC) is a recently rediscovered epigenetic modification of DNA with tissue and cell type specific distribution in mammalian genomes. Recent studies of genomic DNA, found that a substantial fraction of 5-methyl-cytosine (5mC) in CpG dinucleotides is converted to 5hmC and is mainly abundant in the central nerves system. The dynamic nature of this modification implies that 5hmC patterns may exhibit a high degree of cell to cell variation and should be studied by single-cell or single-molecule analysis. We have recently developed a method for optical detection of the epigenetic modification 5hmC at the single-molecule level. We use a chemo-enzymatic reaction to covalently attach a fluorescent reporter molecule to 5hmC nucleotides. Labeled DNA is electrokinetically squeezed into an array of silicon nanochannels and imaged on a fluorescence microscope. This nanochannels array has two entrance points in which the tangled DNA can enter, by high electrical force. The molecules enter to wide nanochannels and are then forced to straighten by nano-pillars which are positioned in the entrance to the narrow nanochannels.
5hmC residues are visible as fluorescent spots along the DNA contour. The human genomic DNA was labeled with an additional color at specific sequence motifs (GCTCTTC) to generate a locus specific pattern of dots that can be used in order to map the molecule onto the reference genome. We show first results of single-molecule epigenetic mapping of chromosomal DNA extracted from human peripheral blood cells. DNA molecules typically spanning several hundred Kbp (and up to 2Mbp) were visualized in the channels.
[501] THE INTERMOLECULAR FORCES THAT GOVERN THE FORMATION OF AMYLOID-LIKE FIBERS IN A BACTERIAL BIOFILM
razan abbasi1
1 Hebrew University of Jerusalem; Givat Ram
Razan Abbasi and Liraz Chai
The School of Chemistry, The Hebrew University of Jerusalem
A biofilm is a group of microorganisms in which cells stick to each other on a surface. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings. The cells in a biofilm are embedded within a self-produced matrix of extracellular polymeric substance (EPS). The EPS is generally composed of extracellular DNA, proteins and polysaccharides. In biofilms of our model organism, the soil bacterium Bacillus subtilis, cells are held together by extracellular cell-anchored amyloid-like fibers that are composed primarily of the protein TasA. TasA has an accessory protein, TapA, that serves both to anchor the fibers to the cell wall and to assemble TasA into fibers. Despite the knowledge of the location of TapA in the biofilm, little is known about the mechanism of action of this protein. In particular, we are interested in studying the TapA-TasA and the TapA – cell surface interactions in order to better understand the role of TapA in the formation of the amyloid-like fibers. A molecular understanding of the assembly of the amyloid-like fibers may lead the way to developing new anti-biofilm drugs.
[522] SILICON NANOWIRES ARRAY FOR MONITORING OF BCTERIAL BIOFILM METABOLIC ACTIVITY
Ella Davidi1, Vadim Krivitsky1, Marina Zverzhinetsky1, Fernando Patolsky1
1 School of Chemistry; School of Chemistry
Silicon Nanowires Array for Monitoring of Bcterial Biofilm Metabolic Activity
Abstract
Bacterial biofilms cause severe infections, which are usually difficult to remove without invasive surgery. Cell metabolism, and in particular glucose metabolism, has been shown to reflect the state of living cells and microorganisms. Our goal is to investigate metabolic activity of biofilms in real time, in order to adjust the proper medical treatment to overcome biofilm infection. We created a platform that enables monitoring the metabolic activity of biofilms based on glucose consumption and other metabolites secretion. Monitoring the metabolic activity of biofilms, could help us finding effective treatment approaches. Here we present a series of experiments, monitoring in real time the Bacillus Subtilis biofilm metabolic activity. The metabolic activity of cells has been researched and monitored in our lab, using silicon nanowires (SiNWs) arrays, configured as a field-effect transistor (FET), that enable real time, label-free detection of biological species. We used surface modification on SiNWs FET, to generate a monolayer of the electroactive 9,10-dihydroxyathracene species, in order to perform direct sensing of glucose. We have managed to monitor the metabolic pathway of Bacillus Subtilis Biofilms from glutamate and glycerol consumption to glucose consumption. In addition, we managed to track the glucose consumption during several repetitive cycles. These results support the novel application of these biosensors for real time monitoring of bio-samples, for both clinical and environmental applications.
Proton conduction in self-assembled Amyloid β peptide fibrils: the effect of peptide side chains.
Ohad Silberbush1, Moran Amit1 and Prof. Nurit Ashkenasy1, 2
1. Department of Materials Engineering, Ben Gurion University of the Negev, Beer- Sheva, Israel
2. The Ilse Katz Institute for Nanoscale Science & Technology, Ben Gurion University of the Negev, Beer- Sheva, Israel
Abstract:
The chemical diversity, ease of synthesis, and self-assembly propensity of peptides make them attractive materials for bioelectronics applications. Taking example from nature, conduction in such bio mimetic materials could be facilitated either by electrons, protons or both. In particular, the presence of hydrogen donating and accepting groups, such as carboxylic acid and amino groups, at the peptide side chains may facilitate affective proton conduction. Aiming at revealing the effect of such side chains on the unexplored proton conduction of self-assembling peptide nanostructures, we present here a systematic study that examines the dependence of protonic conductance on peptide side chains in amyloid β- based peptide fibers.
The peptides used in this work are mutations of the core sequence of the amyloid β protein, AAKLVFF and AAELVFF, with amine and carboxylic acid side chain residue at the third amino acid in the sequence, respectively. The self-assembly of the peptides into fibrous nanostructures with a varying diameter is verified using atomic force microscopy and scanning electron microscopy. Current-voltage measurements reveal an exponential dependence of the conductance on the relative humidity, as expected for proton conduction. Moreover, the conductance was found to be higher for AAELVFF peptide assemblies than for AAKLVFF assembly at each of the measured relative humidity conditions. This behavior is related to the higher mobility of protons (H+) vs. proton holes (OH-). Our findings demonstrate that protonic conduction may be tuned by a proper peptide sequence design. The ability to generate both proton and proton holes may lay the ground to proton based switches and transistor devices.